Registration Dossier

Administrative data

Description of key information

When tested up to and including 25%, Ethylene glycol dibenzoate did not elicit an SI ≥ 3 and was therefore not considered to be a skin sensitizer. It was established that the EC3 value exceeds 25%. The test substance is not classified for skin sensitising properties.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 April 2017 - 02 May 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
in vivo required for non-EU registration
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
July 2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
March 2003
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
dd. 03 Nov 2015
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
Solubility in vehicle: not indicated
Stability in vehicle: not indicated
Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River France, L’Arbresle, France
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx 9 weeks old
- Weight at study initiation: 16.6 to 23.4 g
- Housing: 5 animals together in polycarbonate cages
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap-water, ad libitum
- Acclimation period: 5 days
- Indication of any skin lesions: no; animals were checked before initiation of dosing and any animals considered unsuitable for use in the study were replaced.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 to 22
- Humidity (%): 42 to 47
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 26 April to 01 May 2017
Vehicle:
acetone/olive oil (4:1 v/v)
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
- Concentrations: 25% and 50%
- Systemic toxicity: no signs at 25%; hunched posture in both animals on days 2 and 3 and slight erythema in one animal on day 2.
- Ear thickness measurements: on day 1 and 3 (prior to dosing) and on day 6, using a digital thickness gauge. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values.

MAIN STUDY: Three groups of five animals were treated with one test item concentration per group (5, 10 and 25%). The highest test item concentration was selected from the pre-screen test. One group of five animals was treated with the vehicle.

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: if the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer.

TREATMENT PREPARATION AND ADMINISTRATION:
- Induction (days 1, 2 and 3): both ears were topically treated with 25 μL test item per ear.
- Excision of the nodes (day 6): each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline containing 20 μCi of 3H-methyl thymidine
- Tissue processing for radioactivity (day 6): Following excision of the nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (maze size: 200 μm, diameter: ± 1.5 cm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) and then stored in the refrigerator until the next day.
- Radioactivity measurements (day 7): Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail as the scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter (2800TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

ANALYSIS:
DPM values were calculated for each animal and for each dose group. A Stimulation Index (SI) was calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean.
If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at the testing facility is an appropriate model for testing for contact hypersensitivity
Key result
Parameter:
SI
Value:
1.7
Test group / Remarks:
5% test item concentration group
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
10% test item concentration group
Key result
Parameter:
SI
Value:
1.5
Test group / Remarks:
25% test item concentration group
Key result
Parameter:
EC3
Value:
> 25
Test group / Remarks:
all treatment groups
Cellular proliferation data / Observations:
- No erythema was observed in any of the animals, scaliness was noted in one animal treated at 10% on Day 5 only. Test item remnants were present on the dorsal surface of the ears of all animals at 25% (between Days 2 and 4), which did not hamper scoring of the skin reactions.
- Mean DPM/animal values for the experimental groups treated with test item concentrations 5, 10 and 25% were 429, 242 and 363 DPM, respectively. The mean DPM/animal value for the vehicle control group was 250 DPM.
- No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.
- The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at the test facility is an appropriate model for testing for contact hypersensitivity.

Results of the pre-screen test:

At a 25% test item concentration no signs of systemic toxicity were noted and no erythema was observed. At a 50% test item concentration hunched posture was noted for both animals on Days 2 and 3 and slight erythema was noted for one of the animals on Day 2.

Test item remnants were present on the dorsal surface of the ears of all animals (between Days 1 and 6), which did not hamper scoring of the skin reactions.

Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values.

Based on the tolerability of the test item, the highest test item concentration selected for the main study was a 25% concentration.

Interpretation of results:
GHS criteria not met
Remarks:
Not classified according to Regulation (EC) 1272/2008
Conclusions:
When tested up to and including 25%, Ethylene glycol dibenzoate did not elicit an SI ≥ 3 and was therefore not considered to be a skin sensitizer. It was established that the EC3 value exceeds 25%. The test substance is not classified for skin sensitising properties according to GHS and Regulation (EC) 1272/2008.
Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the outcome of the available study the substance is not classifed as a skin sensitizer.